DNA encoding peptides of IL-2

ABSTRACT

The invention relates to the use as therapeutic agents of IL-2 peptides and derivatives having biological activity and anti-IL-2 antibodies which mimic or modulate the biological activities of IL-2. The invention also relates to DNA sequences encoding the IL-2 peptides, and to methods of using the IL-2 peptides and derivatives and anti-IL-2 antibodies to modulate or mimic or antagonize the biological activities of IL-2 in vivo and to assay for the presence and activity of the IL-2 receptor.

This application is a continuation application of U.S. application Ser.No. 09/116,594, filed Jul. 16, 1998, which is now U.S. Pat. No.6,168,785.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use as therapeutic agents of IL-2,IL-2 peptides and derivatives which act as interleukin-2 (IL-2) agonistsand antagonists, and to methods of modulating IL-2 activity.

2. Discussion of the Background

Interleukin-2 (IL-2) is the main growth factor of T lymphocytes (Thèzeet al. 1996, Immunol. Today 17:481-486). By regulating T helperlymphocyte activity IL-2 increases the humoral and cellular immuneresponses. By stimulating cytotoxic CD8 T cells and NK cells thiscytokine participates to the defense mechanisms against tumors and viralinfections. IL-2 is used in therapy against metastatic melanoma andrenal adenocarcinoma. IL-2 is used in clinical trials in many forms ofcancer (Lotze and Rosenberg 1988, Interleukin 2 as a PharmaclologicReagent. in Interleukin 2, K. A. Smith, Academic Press: p. 237-89). Itis also used in HIV infected patients and leads to a significantincrease in CD4 counts (Kovacs et al. 1996, New Engl. J. of Medicine1350-6).

Human IL-2 is a protein of 133 amino acids (aa) composed of four αhelices connected by loops of various length; its tridimensionalstructure has been established. IL-2R is composed of three chains α, βand γ. IL-2Rα controls the affinity of the receptor. IL-2Rα and IL-2Rγare responsible for IL-2 signal transduction. The different molecularareas of IL-2 interacting with the three chains of the IL-2 R have beendefined. More specifically it has been determined that a helix A as wellas the NH₂ terminal area of IL-2 (residues 1 to 30) control theinteractions IL-2/IL-2Rβ (Eckenberg et al. 1997, Cytokine 9:488-98):IL-2Rβ chain is the most important in IL-2 signaling (Thèze et al.1990).

The effects of human interleukin-2 (IL-2) on its target cells aremediated through specific cell surface receptors (IL-2R) (Taniguchi etal. (1983) Nature 302:305-310; Robb et al. (1984) Proc. Natl. Acad. Sci.USA 81:6486-6490; Smith K A. 1988a. Interleukin-2; Smith K A (1988b)Science 240:1169-1176). IL-2R comprises at least three subunits encodedby different genes (Minami et al. (1993) Annu. Rev. Immunol. 11:245-267;Taniguchi et al. (1993) Cell 73:5-8). The first component to beidentified, IL-2Rα, is a 55 kDa protein that binds IL-2 with a K_(d) of≅10 nM (Uchiyama et al. (1981) J. Immunol. 126:1293-1297; Leonard et al.(1984) Nature 311:626-631). The role of IL-2Rα (Kumar et al. (1987) J.Immunol. 139:3680-3684) and the influence of IL-2 on IL-2Rα geneexpression have been studied (Bismuth et al. (1985) Eur. J. Immunol.15:723-727; Froussard et al. (1991) Mol. Immunol. 28:87-93). The secondIL-2R component, IL-2Rβ is a 75 kDa protein with a largeintracytoplasmic domain (286 aa) (Teshigawara et al. (1987) J. Exp. Med.165:223-238; Hatakeyama et al. (1989) Science 244:551-556; Tsudo et al.(1989) Proc. Natl. Acad. Sci. USA 86:1982-1986). The last component tobe identified, IL-2Rγ, is a 64 kDa protein (Takeshita et al. (1992)Science 257:379-382; Ishii et al. (1994) Int. Immunol. 6:1273-1277).IL-2Rβ and IL-2Rγ belong to the hematopoietin receptor family whereasIL-2Rα belongs to another family of molecules (Thèze J (1994) Eur.Cytokine Netw. 5:353-368). In the mouse system all three chains arerequired to form a functional receptor (Moreau et al. (1995a) J.Immunol. 155:3401-3408; Chastagner et al. (1996) Eur. J. Immunol.26:201-206). In the human system two receptors are functional. Whenassociated, human IL-2Rβ plus IL-2Rγ form an intermediate affinityreceptor with a K_(d) of ≅1 nM, whereas expression of all three chainsleads to the formation of a high affinity IL-2R (K_(d)≅10 pM).

The structure of IL-2 (MacKay D (1992) Science 257:410-413) is composedof a compact core bundle of four antiparallel α helices connected bythree loops (FIG. 1). Some of the interactions between IL-2 and IL-2Rα(Sauv et al. (1991) Proc. Natl. Acad. Sci. USA 88:4636-4640; Wang et al.(1995) Eur. J. Immunol. 25:1212-1216) and IL-2Rγ subunits (Voss et al.(1993) Proc. Natl. Acad. Sci. USA 90:2428-2432; Buchli et al. (1993)Arch. Biochm. Biophys. 307:411-415) have been elucidated, but less isknown concerning IL-2/IL-2Rβ interaction, despite the fact that IL-2Rβchain plays a very critical role in signal transduction (Taniguchi T(1995) Science 268:251-255).

It has been shown that one substitution Asp20 by Lys (mutant D20K)prevents binding to IL-2Rβ (Collins et al. (1988) Proc. Natl. Acad. Sci.USA 85:7709-7713). In a recent report the role of the sequence (Leu17,Leu18, Leu19, Asp20, Leu21) from IL-2α helix A, in IL-2/IL-2Rβinteractions was analyzed by cassette mutagenesis (Berndt et al. (1994)Biochemistry 33:6571-6577). However the data were difficult to interpretsince most of the proteins produced have multiple mutations inside andoutside of the sequence of interest. Only one analog with a singlemutation was studied (L21V). More surprisingly it was reported in thisstudy that deletion of the segment spanning residues 17-31 (Del1) givesa protein with full agonist activity.

IL-2 peptides and derivatives were described in Cytokine (1997)7:488-498, but were not tested in an in vitro system for biochemicalactivity such as cytokine activity, and in particular for IL-2-likeactivity.

In view of the aforementioned deficiencies attendant with the prior artanalysis of IL-2 agonists and antagonists, as well as with methods ofmodulating IL-2 activity therewith, it is clear that there exists a needin the art for the same.

SUMMARY OF THE INVENTION

Accordingly, one object of this inventions to provide compositionshaving an IL-2-like activity and methods for their use as therapeuticagents. The applications of such recombinant, synthetic or hybridpeptides are thus one object of the invention. These compositions aredefined as having the following characteristics: a) containing one ormore peptides at least five amino acids in length; and b) inhibiting ormimicing the binding of α helix A of interleukin-2 (IL-2) to a β subunitof an IL-2 receptor (IL-2R).

Another object of the invention is the use of a purified peptide havingthe following characteristics: a) the peptide is at least five aminoacids in length; b) the peptide binds to a β subunit of an IL-2 receptor(IL-2R); and c) the peptide induces phosphorylation of the β subunit ofthe IL-2R.

A further object of the invention concerns the preparation of theantibodies which recognize the peptides of the invention, and thetherapeutic use of these antibodies.

A further object of the invention is the use of DNA sequences encodingthe peptides of the invention and their derivatives. Such DNA fragmentsare useful for gene therapy among other applications. The use of a DNAof the following sequence

ATG GCT CCG ACG AGC AGC TCC ACC AAG AAA ACC CAG CTC CAG CTC GAA

CAC CTG CTG CTG GAC CTG CAG ATG ATC CTG AAC GGT ATC AAC AAC

(SEQ ID NO:1) is one particular object of the invention.

Yet another object of the invention is to provide a method for detectingthe activity of an IL-2-like peptide, wherein the IL-2 activity ismeasured by the binding of the IL-2R to the peptide having the IL-2agonist or antagonist activity.

A still further object of the invention is the use of compounds whichinhibit the activity of an IL-2R by contacting the IL-2R with an amountof the selected antagonist peptide sufficient to inhibit binding of IL-2to the IL-2R under conditions that allow binding of the peptide to theIL-2R to occur.

Another object of the invention is to provide a method for the selectionof antibodies specific for the purified peptide with IL-2-like activityas defined herein. These monoclonal or polyclonal antibodies can inhibitbinding of IL-2 to the IL-2R under conditions that allow binding of thepeptide to the IL-2R to occur. The therapeutic use of these antibodiesis also a part of the present invention. In particular, these antibodiesspecific for the purified peptides are useful for treating or preventingundesirable immune reactions such as graft rejection or autoimmunedisorders, for example, rheumatoid arthritis.

A still further object of the invention is to provide a method forinducing in a patient the biological effects of IL-2 by administering tothe patient an amount of the agonist peptide of the invention sufficientto induce those biological effects, or by administering a combination ofvarious cytokines and purified peptide. By various cytokines is meantfor example IL-4, IL-9, IL-15 or IL-2.

Another object of the invention relates to the nucleic acid sequencescorresponding to the amino acid sequence of the purified peptide and itsderivatives according to the invention. A preferred embodiment is thenucleotide sequence encoding the purified peptide IP130 having thefollowing sequence (SED ID NO:2):

Met Ala Pro Tbr Ser Ser Ser Thr Lys Lys Thr Gln eu Gln Leu Glu

His Leu Leu Lea Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn (SEQ ID NO:2) This sequence or a sequence derived therefrom can be inserted in anappropriate vector capable of expressing the product in vivo, in abacterium or in a eukaryotic cell, particularly in yeast or a mammaliancell. These constructs are useful for gene therapy among other uses.

With the foregoing and other objects, advantages and features of theinvention that will become hereinafter apparent, the nature of theinvention may be more clearly understood by reference to the followingdetailed description of the preferred embodiments of the invention andto the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 shows a schematic representation of the human IL-2 structure. Theprotein contains 133 amino-acid residues (molecular weight: 15-18 kDa,depending on the degree of glycosylation). Four α-helices, denoted A(residue positions 6-29), B—B′ (positions 53-72), C (positions 81-97),and D (positions 113-133) surround a central hydrophobic core. ResiduesLeu17 and Asp20 (see text) occur in the N-terminal helix A. Thestructure of the loop between α helix C and α helix D was undetermined.Atomic coordinates were obtained from the Brookhaven Protein Data Bank(Bernstein et al. (1977) J. Mol. Biol. 112:535-542) entry link,deposited by D. B. McKay (MacKay D (1992) Science 257:410-413). Thefigure was drawn with the program Molscript (Kraulis (1991) J. Appl.Crystallogr. 24:946-950).

FIG. 2 shows the binding and binding inhibition of mAb H2-8 which isdeposited at the CNCM under the accession number I-2338 on Oct. 21,1999. Binding experiments: plates were coated with IL-2, peptide 1-22 orpeptide 1-30. Control is represented by non coated plates. Binding ofmAb was revealed with alkaline phosphatase goat anti-mouse polyvalent Igconjugate. Inhibition experiments: concentrations of mAbs H2-8 or 19B11giving ½ maximal binding on IL-2 coated plates were used. Thesedilutions were mixed for 1 hr at 37° C. with the indicated concentrationof inhibitors before addition to wells coated by IL-2.

FIG. 3 shows the binding of mAb H2-8 on peptide 1-30. Plates were coatedwith mAb H2-8 and were incubated with peptide 1-30 as described.

FIG. 4 shows the biological effects of mAb H2-8 on IL-2 Pro¹²⁵.Different concentrations of IL-2 Pro¹²⁵ were tested on the proliferationof TS1β cells (IL-2Rα⁻, human IL-2Rβ⁺, mouse IL-2Rγ⁺). The proliferationwas measured by [^(3H)TdR] incorporation as indicated.

Various concentrations (indicated in parenthesis—μg/ml) of mAb H2-8 weretested. As control the absence of effects of mAb H2-8 on IL-2 dependentproliferation of TS1β cells was verified.

FIG. 5 shows the model of IL-2/IL-2R interactions. FIG. 8(A) Position ofresidues Leu17 and Asp20 in the IL-2 structure with respect to helicesA, C and D, in a view perpendicular to the axis of helix A. FIG. 8(B)Position of residue Leu17 with respect to helices A and B-B′. The sidechain of Leu17 is located in a leucine-rich hydrophobic core of themolecule. The charged side chain of Asp20 is partly exposed to solvent.The two orientations of the molecule shown here are roughlyperpendicular to that shown in FIG. 1. FIG. 8(C) The model of theIL-2/IL-2R complex (Bamborough et al. (1994) Structure 2:839-851) isbased on the structure of the human growth hormone and its receptor (deVos et al. (1992) Science 255:306-312). For clarity, only α-helices Aand D of IL-2 and the β and γ chains of the receptor are shown. The IL-2positions studied, Leu17, Asp20 and residues Arg15 and Tyr134 of IL-2Rβare labelled. Positions Cys125 and Ser127 are also shown. Secondarystructural elements as defined by the program DSSP (Kabsch et al. (1983)Biopolymers 22:2577-2637). Atomic coordinates of the complex wereobtained from the Brookhaven Protein Data Bank, entry code film.

FIG. 6 (SEQ ID NO:3) is a schematic representation of IL-2 (SEQ ID NO:1)/IL-2R interactions and IP130 (SEQ ID NO:2). IL-2 receptor is composedof three subunits (α, β, γ) (See Imm. Today, 1996).

FIG. 7 demonstrates that IP 130 induces proliferation and acts insynergy with IL-2. Proliferative activity was tested on TS1βP2 (grown inIL-2). Background of 1.4×10³ cpm was subtracted. Synergy with IL-2 isalso observed. TS1β target cells are derived from TS1 murine cells(which only express murine IL-2Rγ), after transfection with the humanIL-2Rγ gene.

FIG. 8 shows that the proliferation induced by IP130 is not due tosynergy with residual growth factor coming from the culture medium. TS1βcells, used in this study, are cultured in IL-4. TS1β proliferates withIP130 and this proliferation is not inhibited by 11B11 mAB, whichneutralizes proliferation induced by IL-4. FIG. 8A shows IP130proliferative activity. FIG. 8B shows IL-4 proliferative activity. FIG.8C shows IP130 +mAB 11B11 (anti-IL-4). FIG. 8D shows IL-4+mAB 11B11 (□)and IL-4+mAB 145 (control mAB) (∇). Proliferative activity was tested onTS1β4 (grown in IL-4).

FIG. 9 demonstrates that human IL-2Rβ is essential for the proliferationinduced by IP130. FIG. 12A shows IP130 activity on TS1 cells transfectedwith human IL-2R β. FIG. 12B shows the effect of anti-human IL-2Rβneutralizing antibody (A41) on IP130 activity. TS1 cells onlyproliferate after transfection by the human IL-2Rβ gene. As for IL-2,murine IL-2Rβ chain does not allow proliferation in the presence ofIP130. TS1β proliferation induced by IP130 is specifically neutralizedby the mAB A41 (anti human IL-2Rβ).

FIG. 10 is a summary of IP130 (SEQ ID NO:2) and of derivative molecule'sstructure-function studies. A family of peptides was studied forhelicity, oligomerization and biologic activity (see also generalpresentation of the data).

FIG. 11 is a model of IP130/IL-2Rβ interactions. IP130 is tetrameric andIL-2Rβ forms a dimer in solution. Proposed from the results obtainedwith the three dimensional structure of IL-2 and growth hormone/receptorcomplex.

FIG. 12 shows the pattern of phosphorylation induced by IP130 andinvolvement of SHC. IP130 induces SHC protein phosphorylation. The twobands corresponding to SHC isoforms are phosphorylated after a tenminute stimulation by IP130. Kinetics of Shc phosphorylation is shown onthe left. A Western blot of Shc protein is shown on the right.

FIG. 13 is an electrophoretic mobility shift assay which shows thatIP130 does not induce STAT activity. STAT activation is analyzed inKIT225 cells nuclear extracts after stimulation by IL-2, IP130 orIL-2+IP130. Only cells stimulated by IL-2 or IL-2+IP130 show a STATactivation. β-casein probes were used in the study. The same resultswere obtained with two other probes (GAS and GIRE).

FIG. 14 depicts a model of signal transduction and IP130. IL-2 usesthree main pathways: 1°/JAK/STAT depending on IL-2Rβγ complex;2°/SHC/MAPK initiated on the phosphorylated IL-2 Rβ chain and 3°/IP3K.In accordance with the (IP130)₄/(IL-2Rβ)₂ model (FIG. 14), IP130 doesnot induce the JAK/STAT pathway but induces the SHC/MAPK pathway. (JAK:janus activated kinase; PI3K: phosphatidyl inositol, 3-phosphate kinase;STAT: signal transducers and activators of transcription; MAPKK: mitogenactivated protein kinase; MAPK: mitogen activated protein kinase; :induction of transcription).

FIG. 15 shows the cell cycle entry (S+G2/M) of IP130 stimulated NKcells. PBMC are stimulated by IL-2, IP130 or IL-2+IP130. Non-specificresponses (J+1 in medium) are subtracted. NK cells entry into S+G2/Mphases are measured by propidium iodide and analyzed with the ModFit 2.0software (Becton Dickinson).

FIG. 16 shows that IP130 stimulates LAK activity. In this experiment,the kinetics of LAK activity stimulation has been studied. Histogramsshow the results with an effector/target ratio of ten. Δ% lysis=lysisinduced by IL-2 and/or IP130-spontaneous lysis.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to the use of IL-2 peptides derived frominterleukin-2 (IL-2), for their therapeutic use in mammals, andparticularly in humans. The peptides are selected from fragments of IL-2and derivatives of IL-2. The derivatives are defined as containing anamino acid sequence capable of binding to the IL-2R β chain under theconditions described herein, or capable of binding to the monoclonalantibodies produced by H2-8 hybridoma. The invention also relates toantibodies directed against the peptides according to the inventionwhich likewise mimic and/or modulate IL-2 activity. The diagnostic andtherapeutic approaches involve the use of the purified peptides and theantibodies for detecting and/or modulating IL-2 binding to IL-2R invitro and in vivo.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

The terms “IL-2 peptide,” “IL-2 agonist/antagonist,” “IP130/IP130derivatives,” and any variants not specifically listed, may be usedherein interchangeably, and as used throughout the present application.These terms refer to proteinaceous material including single or multipleproteins or recombinant product or peptides obtained by chemicalsynthesis, and extend to those proteins having the amino acid sequencedata described herein and presented in FIG. 9 (SEQ ID NO: 1). Theprofile of biological activities set forth hereinafter is one of theaspects of the present application. Accordingly, proteins displayingsubstantially equivalent or altered activity are likewise contemplated.These modifications may be deliberate, for example, such asmodifications obtained through site-directed mutagenesis, or may beaccidental, such as those obtained through mutations in hosts that areproducers of the complex or its named subunits. Also, the terms “IL-2peptide,” “IL-2 agonist/antagonist” and “IP130/IP130 derivatives orpurified peptide(s)” are intended to include within their scope proteinsspecifically recited herein as well as all substantially homologousanalogs and allelic variations.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property or immunoglobulin-binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969),abbreviations for amino acid residues are shown in the following Tableof Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn asparagine C Cys cysteine

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The above Table ispresented to correlate the three-letter and one-letter notations whichmay appear alternately herein.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” is defined as a molecule comprised of two ormore ribonucleotides, preferably more than three. Its exact size willdepend upon many factors which, in turn, depend upon the ultimatefunction and use of the oligonucleotide.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

It should be appreciated that also within the scope of the presentinvention are the biological uses of the DNA sequences encoding IL-2peptides having the same amino acid sequence as IP130 (SEQ ID NO:2), butwhich are degenerate to the DNA encoding SEQ ID NO:2. By “degenerate to”is meant that a different three-letter codon is used to specify aparticular amino acid. It is well known in the art that the followingcodons can be used interchangeably to code for each specific amino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUUor CUC or CUA or CUG Isoleucine (Ile or I) AUU or AUC or AUA Methionine(Met or M) AUG Valine (Val or V) GUU or GUC of GUA or GUG Serine (Ser orS) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCCor CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Alaor A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y) UAU or UAC Histidine(His or H) CAU or CAC Glutamine (Gln or Q) CAA or CAG Asparagine (Asn orN) AAU or AAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAUor GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU orUGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine(Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGGTermination codon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

Modifications of the peptides can be made in the DNA encoding SEQ IDNO:2 such that a particular codon is changed to a codon which codes fora different amino acid. Such a mutation is generally made by making thefewest nucleotide changes possible. A substitution mutation of this sortcan be made to change an amino acid in the resulting protein in anon-conservative manner (i.e., by changing the codon from an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic to an amino acid belonging to another grouping) or in aconservative manner (i.e., by changing the codon from an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic to an amino acid belonging to the same grouping). Such aconservative change generally leads to less change in the structure andfunction of the resulting protein. A non-conservative change is morelikely to alter the structure, activity or function of the resultingprotein. The present invention should be considered to include sequencescontaining conservative changes which do not significantly alter theactivity or binding characteristics of the resulting protein.

The following is one example of various groupings of amino acids:

Amino acids with nonpolar R groups Alanine Valine Leucine IsoleucineProline Phenylalanine Tryptophan Methionine Amino acids with unchargedpolar R groups Glycine Serine Threonine Cysteine Tyrosine AsparagineGlutamine Amino acids with charged polar R groups (negatively charged atPh 6.0) Aspartic acid Glutamic acid Basic amino acids (positivelycharged at pH 6.0) Lysine Arginine Histidine (at pH 6.0) Anothergrouping may be those amino acids with phenyl groups: PhenylalanineTryptophan Tyrosine Another grouping may be according to molecularweight (i.e., size of R groups): Glycine 75 Alanine 89 Serine 105Proline 115 Valine 117 Threonine 119 Cysteine 121 Leucine 131 Isoleucine131 Asparagine 132 Aspartic acid 133 Glutamine 146 Lysine 146 Glutamicacid 147 Methionine 149 Histidine (at pH 6.0) 155 Phenylalanine 165Arginine 174 Tyrosine 181 Tryptophan 204

Particularly preferred conservative substitutions are:

Lys for Arg and vice versa such that a positive charge may bemaintained;

Glu for Asp and vice versa such that a negative charge may bemaintained;

Ser for Thr such that a free —OH can be maintained; and

Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced in IL-2 or peptidesthereof to substitute an amino acid with a particularly preferableproperty. For example, a Cys may be introduced a potential site fordisulfide bridges with another Cys. A His may be introduced as aparticularly “catalytic” site (i.e., His can act as an acid or base andis the most common amino acid in biochemical catalysis). Pro may beintroduced because of its particularly planar structure, which inducesβ-turns in the protein's structure.

The biologically active peptides of the invention preferably encompass aregion of the IL-2 sequence which includes amino acids 17-20, althoughone or more of these amino acid residues may be substituted with anotheramino acid, or a modified amino acid. The use of the preferred peptidecontaining at least 5 amino acids in length, more preferably 8-12 aminoacids, and most preferably at least 15 amino acids in length is oneaspect of the invention, as well as the use of the peptide of theinvention based on amino acids 1-30 of IL-2.

Biological or physiological activity of IL-2 may be considered toinclude the stimulation of CD4, CD8 and NK cells, and may includeantiviral and antitumor activities. Biological or physiological activityof IP130 and other peptides of the invention may include the foregoingactivities of IL-2, as well as induction of SHC phosphorylation andinduction of the SHC/MAPK pathway.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies, the last mentioned described infurther detail in U.S. Pat. Nos. 4,816,397 and 4,816,567.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

Fab and F(ab′)₂ portions of antibody molecules are prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced fromF(ab′)₂ portions followed by reduction of the disulfide bonds linkingthe two heavy chain portions as with mercaptoethanol, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact antibody molecules ispreferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

Preferred antibodies of the present invention bind to the peptides ofthe invention, described above. The antibodies which bind the peptidesof the invention may be used as diagnostic agents for analyzing IL-2binding to its receptor, and may also be used as therapeutic agents, toenhance or inhibit the binding of IL-2 to its receptor. In a preferredembodiment, the antibody of the invention inhibits the binding of IL-2and/or IP130 to the IL-2R.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to prevent, and preferably reduce by at least about 30percent, more preferably by at least 50 percent, most preferably by atleast 90 percent, a clinically significant change in a feature ofpathology such as for example, elevated blood pressure, fever or whitecell count as may attend its presence and activity.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20° C. below the predicted or determined T_(m) with washes of higherstringency, if desired.

In its primary aspect, the present invention concerns the use of IL-2peptides which modulate IL-2 activity. By “modulate” is meant eitheragonist or antagonist activity which either increases or suppresses thephysiological effects of IL-2, such as the proliferation of cells, asdescribed below.

As stated above, the present invention also relates to a recombinant DNAmolecule or cloned gene, or a degenerate variant thereof, which encodesan IL-2 peptide, or a fragment thereof, that possesses a molecularweight preferably of about 2-5 kD and an amino acid sequence set forthin FIG. 9 (SEQ ID NO:2) or a sequence wherein SEQ ID NO:2 is modified byinsertion, deletion and/or substitution; preferably a nucleic acidmolecule, in particular a recombinant DNA molecule or cloned gene,encoding the peptide has a nucleotide sequence, is complementary to, orhybridizes under standard hybridization conditions to a DNA sequenceencoding SEQ ID NO:2.

The possibilities both diagnostic and therapeutic that are raised by theexistence of the IL-2 peptides, derive from the fact that the peptidesappear to participate in direct and causal protein-protein interactionbetween the IL-2 peptide and the IL-2 receptor, specifically IL-2Rβ, andthose factors that thereafter mediate cellular events. In particular theIP130 peptide has been shown to induce phosphorylation of IL-2Rβ, toinduce c-myc; and to induce natural killer (NK) cells to enter the cellcycle. As suggested earlier and elaborated further on herein, thepresent invention contemplates pharmaceutical intervention in thecascade of reactions in which the IL-2R is implicated, to modulate theactivity initiated by IL-2 and peptides thereof.

Thus, in instances where it is desired to reduce or inhibit the IL-2induced activity, an appropriate IL-2 peptide inhibitor of IL-2 could beintroduced to block the interaction of the IL-2 with the IL-2R.Correspondingly, instances where insufficient IL-2 induced activity istaking place could be remedied by the introduction of additionalquantities of the appropriate IL-2 peptide agonist, such as IP130, orits chemical or pharmaceutical cognates, analogs, fragments and thelike.

As discussed earlier, the IL-2 peptides or their binding partners orother ligands or agents exhibiting either mimicry or antagonism to IL-2or control over its production, may be prepared in pharmaceuticalcompositions, with a suitable carrier and at a strength effective foradministration by various means to a patient experiencing an adversemedical condition associated with undesirable levels of IL-2 for thetreatment thereof. A variety of administrative techniques may beutilized, among them parenteral techniques such as subcutaneous,intravenous and intraperitoneal injections, catheterizations and thelike. Average quantities of the IL-2 peptides or their subunits may varyand in particular should be based upon the recommendations andprescription of a qualified physician or veterinarian.

Also, antibodies including both polyclonal and monoclonal antibodies,and drugs that modulate the production or activity of IL-2 and/orpeptides thereof may possess certain diagnostic applications and may forexample, be utilized for the purpose of detecting and/or measuring IL-2receptor activity or the like. For example, IL-2 or peptides thereof maybe used to produce both polyclonal and monoclonal antibodies tothemselves in a variety of cellular media, by known techniques such asthe hybridoma technique utilizing, for example, fused mouse spleenlymphocytes and myeloma cells. Likewise, small molecules that mimic orantagonize the activity(ies) of the IL-2 peptides of the invention maybe discovered or synthesized, and may be used in diagnostic and/ortherapeutic protocols.

The general methodology for making monoclonal antibodies by hybridomasis well known. Immortal, antibody-producing cell lines can also becreated by techniques other than fusion, such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980);Hammerling et al., “Monoclonal Antibodies And T-cell Hybridomas” (1981);Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; 4,493,890.

Panels of monoclonal antibodies produced against IL-2 peptides can bescreened for various properties; i.e., isotype, epitope, affinity, etc.Of particular interest are monoclonal antibodies that modulate theactivity of IL-2 or peptides thereof. Such monoclonals can be readilyidentified in cellular proliferation assays. High affinity antibodiesare also useful when immunoaffinity purification of native orrecombinant IL-2 or IL-2 peptides is possible.

Preferably, the anti-IL-2 antibody used in the diagnostic methods ofthis invention is an affinity purified polyclonal antibody. Morepreferably, the antibody is a monoclonal antibody (mAb). In addition, itis preferable for the anti-IL-2 antibody molecules used herein be in theform of Fab, Fab′, F(ab′)₂ or F(v) portions of whole antibody molecules.

A diagnostic method of the present invention comprises examining acellular sample or medium by means of an assay including an effectiveamount of a labeled IL-2 peptide or an antagonist thereof, such as ananti-IP130 antibody, preferably an affinity-purified polyclonalantibody, and more preferably a mAb. In addition, it is preferable forthe anti-IL-2 antibody molecules used herein be in the form of Fab,Fab′, F(ab′)₂ or F(v) portions or whole antibody molecules. Aspreviously discussed, patients capable of benefitting from this methodinclude those suffering from cancer, a pre-cancerous lesion, a viralinfection or other like pathological derangement. Methods for isolatingthe IL-2 peptide and inducing anti-IL-2 antibodies and for determiningand optimizing the ability of anti-IL-2 antibodies to assist in theexamination of the target cells are all well-known in the art.

Methods for producing polyclonal anti-polypeptide antibodies arewell-known in the art. See U.S. Pat. No. 4,493,795 to Nestor et al. Amonoclonal antibody, typically containing Fab and/or F(ab′)₂ portions ofuseful antibody molecules, can be prepared using the hybridomatechnology described in Antibodies—A Laboratory Manual, Harlow and Lane,eds., Cold Spring Harbor Laboratory, N.Y. (1988), which is incorporatedherein by reference. Briefly, to form the hybridoma from which themonoclonal antibody composition is produced, a myeloma or otherself-perpetuating cell line is fused with lymphocytes obtained from thespleen of a mammal hyperimmunized with an IL-2 peptide or IL-2 R-bindingportion thereof.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 6000. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody useful in practicingthis invention are identified by their ability to immunoreact with thepresent IL-2 mutant or peptide and their ability to inhibit specifiedIL-2 activity in target cells.

A monoclonal antibody useful in practicing the present invention can beproduced by initiating a monoclonal hybridoma culture comprising anutrient medium containing a hybridoma that secretes antibody moleculesof the appropriate antigen specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well-known techniques.

Media useful for the preparation of these compositions are bothwell-known in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol.8:396 (1959)) supplemented with 4.5 gm/l glucose, 20 mm glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Methods for producing monoclonal anti-IL-2 antibodies are alsowell-known in the art. See Niman et al., Proc. Natl. Acad Sci. USA,80:4949-4953 (1983). Typically, the present IL-2 peptide or a peptideanalog is used either alone or conjugated to an immunogenic carrier, asthe immunogen in the before described procedure for producing anti-IL-2monoclonal antibodies. The hybridomas are screened for the ability toproduce an antibody that immunoreacts with the IL-2 mutant or peptideanalog.

The present invention further contemplates the use of therapeuticcompositions which are useful in practicing the therapeutic methods ofthis invention. In one embodiment, the therapeutic composition includes,in admixture, a pharmaceutically acceptable excipient (carrier) and oneor more of a IL-2 peptide, a purified peptide or a derivative thereof ora polypeptide analog thereof or fragment thereof, as described herein asan active ingredient. In a preferred embodiment, the therapeuticcomposition comprises an active compound containing a purified peptidecapable of modulating the specific binding of the present IL-2 with theIL-2R.

The preparation of therapeutic compositions which contain polypeptides,analogs or active fragments as active ingredients is well understood inthe art. Typically, such compositions are prepared as injectables,either as liquid solutions or suspensions, however, solid forms suitablefor solution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thecomposition can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents which enhance theeffectiveness of the active ingredient.

The use of the compositions may be by administration in a mannercompatible with the dosage formulation, and in a therapeuticallyeffective amount. The quantity to be administered depends on the subjectto be treated, capacity of the subject's immune system to utilize theactive ingredient, and degree of modulation of IL-2 binding capacitydesired. Precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner and are peculiarto each individual. However, suitable dosages may range from about 0.1to 20, preferably about 0.5 to about 10, and more preferably one toseveral, milligrams of active ingredient per kilogram body weight ofindividual per day and depend on the route of administration. Suitableregimes for initial administration and booster shots are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations of ten nanomolar to ten micromolarin the blood are contemplated.

The use of the therapeutic compositions may be by administration in acomposition which further includes an effective amount of the IL-2agonist/antagonist or analog thereof, and one or more of the followingactive ingredients: an antibiotic, a steroid.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg”mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml”means milliliter, “l” means liter.

Another feature of this invention is the expression of the DNA sequencesdisclosed herein. As is well known in the art, DNA sequences may beexpressed by operatively linking them to an expression control sequencein an appropriate expression vector and employing that expression vectorto transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. Coli plasmidscol El , pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAS, e.g., the numerous derivatives of phage λ, e.g., NM989, andother phage DNA, e.g., M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2 μ plasmid or derivatives thereof; vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage λ, the control regionsof fd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast α-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, R1.1, B-W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences of this invention onfermentation or in large scale animal culture.

It is further intended that for the therapeutic use of the peptidesaccording to the invention, the IL-2 peptide analogs may be preparedfrom nucleotide sequences of the protein complex/subunit derived withinthe scope of the present invention. Analogs, such as fragments, may beproduced, for example, by pepsin digestion of IL-2 material. Otheranalogs, such as muteins, can be produced by standard site-directedmutagenesis of IL-2 coding sequences. Analogs exhibiting “IL-2 activity”such as small molecules, whether functioning as promoters or inhibitors,may be identified by known in vivo and/or in vitro assays.

As mentioned above, a DNA sequence encoding IL-2 peptides can beprepared synthetically rather than cloned. The DNA sequence can bedesigned with the appropriate codons for the IL-2 peptide amino acidsequence. In general, one will select preferred codons for the intendedhost if the sequence will be used for expression. The complete sequenceis assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence. See, e.g., Edge,Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay etal., J. Biol. Chem., 259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes whichwill express IL-2 peptide analogs or “muteins”. Alternatively, DNAencoding muteins can be made by site-directed mutagenesis of native IL-2genes or cDNAs, and muteins can be made directly using conventionalpolypeptide synthesis.

A general method for site-specific incorporation of unnatural aminoacids into proteins is described in Christopher J. Noren, Spencer J.Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science,244:182-188 (April 1989). This method may be used to create analogs withunnatural amino acids.

In accordance with the gene therapy applications of the presentinvention, the preparation of antisense oligonucleotides and ribozymesmay be used to modulate the expression of IL-2 at the translationallevel. This approach utilizes antisense nucleic acid and ribozymes toblock translation of a specific mRNA, either by masking that mRNA withan antisense nucleic acid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule. (See Weintraub, 1990;Marcus-Sekura, 1988.) In the cell, they hybridize to that mRNA, forminga double stranded molecule. The cell does not translate an mRNA in thisdouble-stranded form. Therefore, antisense nucleic acids interfere withthe expression of mRNA into protein. Oligomers of about fifteennucleotides and molecules that hybridize to the AUG initiation codonwill be particularly efficient, since they are easy to synthesize andare likely to pose fewer problems than larger molecules when introducingthem into cells. Antisense methods have been used to inhibit theexpression of many genes in vitro (Marcus-Sekura, 1988; Hambor et al.,1988).

The DNA sequences described herein may thus be used to prepare antisensemolecules against, and ribozymes that cleave mRNAs for IL-2 or moleculeswhich stimulate or reduce IL-2 secretion and their ligands.

The present invention also relates to a variety of diagnosticapplications, including methods for detecting IL-2 presence andactivity, by reference to their ability to elicit the activities whichare mediated by the present IL-2 peptides. As mentioned earlier, theIL-2 peptide can be used to produce antibodies to itself by a variety ofknown techniques, and such antibodies could then be isolated andutilized as in tests for the presence of particular IL-2 and/or IL-2 Ractivity in suspect target cells.

As described in detail above, antibody(ies) to the IL-2 peptides can beproduced and isolated by standard methods including the well knownhybridoma techniques. For convenience, the antibody(ies) to the IL-2peptide will be referred to herein as Ab₁ and antibody(ies) raised inanother species as Ab₂.

The presence of IL-2 and IL-2 peptides in cells can be ascertained bythe usual immunological procedures applicable to such determinations. Anumber of useful procedures are known. Three such procedures which areespecially useful utilize either the IL-2 peptide labeled with adetectable label, antibody Ab₁ labeled with a detectable label, orantibody Ab₂ labeled with a detectable label. The procedures may besummarized by the following equations wherein the asterisk indicatesthat the particle is labeled, and “ILP” stands for the IL-2 peptide:

A. ILP*+Ab₁=ILP*Ab₁

B. ILP+Ab*=ILPAb₁*

C. ILP+Ab₁+Ab₂*=ILPAb₁Ab₂*

The procedures and their application are all familiar to those skilledin the art and accordingly may be utilized within the scope of thepresent invention. The “competitive” procedure, Procedure A, isdescribed in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure C, the“sandwich” procedure, is described in U.S. Pat. Nos. RE 31,006 and4,016,043. Still other procedures are known such as the “doubleantibody,” or “DASP” procedure.

In each instance, the ILP forms complexes with one or more antibody(ies)or binding partners and one member of the complex is labeled with adetectable label. The fact that a complex has formed and, if desired,the amount thereof, can be determined by known methods applicable to thedetection of labels.

It will be seen from the above, that a characteristic property of Ab₂ isthat it will react with Ab₁. This is because Ab₁ raised in one mammalianspecies has been used in another species as an antigen to raise theantibody Ab₂. For example, Ab₂ may be raised in goats using rabbitantibodies as antigens. Ab₂ therefore would be anti-rabbit antibodyraised in goats. For purposes of this description and claims, Ab₁ willbe referred to as a primary or anti-ILP antibody, and Ab₂ will bereferred to as a secondary or anti-Ab₁ antibody.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example, fluorescein, rhodamine, auramine,Texas Red, AMCA blue and Lucifer Yellow. A particular detecting materialis anti-rabbit antibody prepared in goats and conjugated withfluorescein through an isothiocyanate.

The IL-2 peptide or its binding partner(s) can also be labeled with aradioactive element or with an enzyme. The radioactive label can bedetected by any of the currently available counting procedures. Thepreferred isotope may be selected from ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr,⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

A particular assay system which can be utilized in accordance with thepresent invention, is known as a receptor assay. In a receptor assay,the material to be assayed is appropriately labeled and then certaincellular test colonies are inoculated with a quantity of both thelabeled and unlabeled material after which binding studies are conductedto determine the extent to which the labeled material binds to the cellreceptors. In this way, differences in affinity between materials can beascertained.

A further embodiment of this invention is the diagnostic application ofcommercial test kits suitable for use by a medical specialist. The kitmay be prepared to determine the presence or absence of predeterminedIL-2R activity or predetermined IL-2 activity capability in suspectedtarget cells. In accordance with the testing techniques discussed above,one class of such kits will contain at least the labeled IL-2 peptide orits binding partner, for instance an antibody specific thereto, anddirections, of course, depending upon the method selected, e.g.,“competitive,” “sandwich,” “DASP” and the like. The kits may alsocontain peripheral reagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of thepresence or capability of cells for predetermined IL-2 R activity,comprising:

(a) a predetermined amount of at least one labeled immunochemicallyreactive component obtained by the direct or indirect attachment of thepresent IL-2 peptide factor or a specific binding partner thereto, to adetectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

(a) a known amount of the IL-2 peptide as described above (or a bindingpartner) generally bound to a solid phase to form an immunosorbent, orin the alternative, bound to a suitable tag, or plural such endproducts, etc. (or their binding partners) one of each;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

In a further variation, the test kit may be prepared and used for thepurposes stated above, which operates according to a predeterminedprotocol (e.g. “competitive,” “sandwich,” “double antibody,” etc.), andcomprises:

(a) a labeled component which has been obtained by coupling the I1-2peptide to a detectable label;

(b) one or more additional immunochemical reagents of which at least onereagent is a ligand or an immobilized ligand, which ligand is selectedfrom the group consisting of:

(i) a ligand capable of binding with the labeled component (a);

(ii) a ligand capable of binding with a binding partner of the labeledcomponent (a);

(iii) a ligand capable of binding with at least one of the component(s)to be determined; and

(iv) a ligand capable of binding with at least one of the bindingpartners of at least one of the component(s) to be determined; and

(c) directions for the performance of a protocol for the detectionand/or determination of one or more components of an immunochemicalreaction between the IL-2 peptide and a specific binding partnerthereto.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLES Example 1

Characterization of Mouse Monoclonal Antibody H2.8

Female BALB/c mice were repeatedly immunized with 25 to 50 μg of peptide1-30 per injection. The peptide was coupled to the KLH carrier andinjected with Complete Freund's adjuvant (first injection) or incompleteFreund's adjuvant (subsequent injections). The titer of the anti-IL-2activity was assessed in a group of five animals. Spleen cells from theanimal giving the best response were used for fusion with cell lineSP2-0. Four hybridomas with specific anti-IL-2 activity were cloned. ThemAbs were purified from the corresponding ascitic fluid by amoniumsulfate precipitation. The purity of the reagents (>80%) was verified bypolyacrylamide gels. The properties of the mAbs were characterized. Theresults are reported only for mAb H2-8. The isotype (IgG1) and the Kd(1.4×10⁻⁹M) of mAb H2-8 were determined.

Mouse mAbs 19B11 (IgG1) and 2C4 (IgG1) previously characterized (Moreauet al (1995b) Mol. Immunol. 32:1047-1056; Rebollo et al (1992) Mol.Immunol. 29:119-130) were used as controls. mAbs 19B11 and 2C4 inhibitthe binding of IL-2 to IL-2Rβ and recognize the peptides 1-10 (seebelow), 1-22 and 1-30. Rat monoclonal 11B11 (IgG, k) specific for murineIL-4 was provided by Dr. W. Paul (National Institute of Health, BethesdaMd., USA and used as previously described (Moreau et al. (1995b) Mol.Immunol. 32:1047-1056).

The inhibitory effect of purified mAb H2-8 was first assayed on thebinding of ¹²⁵I-labeled IL-2. Its effects were measured on twotransfectants derived from a mouse cell line expressing only mIL-2Rγ.Transfectant TS1β and TS1α were obtained after transfection with humanIL-2Rβ and human IL-2Rα cDNA, respectively. Both cell lines bind IL-2.mAb H2-8 inhibits the binding of IL-2 to TS1β without significantlyaffecting IL-2 binding to TS1α (data not shown).

The binding properties of mAb H2-8 were studied by ELISA. Plates werecoated with either IL-2 or peptides 1-22 or 1-30. mAb H2-8 binds to IL-2and peptide 1—30, but does not recognize peptide 1-22. As control mAb19B11 (previously characterized) recognized both peptides (FIG. 2).

Binding inhibition experiments were performed to further characterizethe specificity of mAb H2-8. Plates were coated with IL-2 and aconcentration of mAb H2-8 giving approximatively 50% of maximum bindingwas used. H2-8 was preincubated with different peptides including fivedecapeptides (1-10, 5-15, 10-20, 15-25, 20-30). Only IL-2 and peptide1-30 were able to inhibit the binding of mAb H2-8 to IL-2. Peptide 1-30was the most efficient inhibitor in these experiments (FIG. 2). Thisresult is compatible with the fact that isolated peptide 1-30 folds inan α helical configuration (α-helix content of 50%±7%) whereas peptide1-22 does not (13%±5%) as measured by circular dichroism. Thereforepeptide 1-30 may adopt some unique structural conformation very close tothat of native IL-2. As control the binding of mAb 19B11 is inhibited byIL-2 but also by peptides 1-10, 1-22 and 1-30. This confirms that theepitope of mAb 19B11 is near the NH₂ terminal position of IL-2 aspreviously suggested (Moreau et al. (1995b) Mol. Immunol. 32:1047-1056).

Example 2

Peptide Synthesis

Peptides were synthesized by the stepwise solid-phase using theBoc/trifluoroacetic acid method (Merrifield (1963) J. Am. Chem. Soc.85:2149-2145), on a p-methyl-benzhydrylamine resin (Applied Biosystems)with an Applied Biosystems 430A peptide synthesizer.

Example 3

Involvement of aa at Positions 17 and 20 in mAb H2-8 Recognition

The reactivity of mAb H2-8 to various IL-2 mutants including one mutantat position 17 (Leu→Asp), four mutants at position 20 (Asp→Asn; Asp→Lys;Asp→Arg and Asp→Leu) and a double mutant 17-20 (Leu17→Asp and Asp20→Leu)were tested by Western blot analysis (FIG. 3).

mAb H2-8 does not recognize mutations at position 20 or the doublemutant (Thèze J (1994) Eur. Cytokine Netw. 5:353-368; Moreau et al.(1995a) J. Immunol. 155:3401-3408; Chastagner et al. (1996) Eur. J.Immunol. 26:201-206; MacKay D (1992) Science 257:410-413). Recognitionof the mutation at position 17 is also affected. As positive control theresults obtained with mAb 2C4 that recognize an epitope near the NH₂terminal area of IL-2 are shown. Since this mAb (as 19B11) recognizespeptide 1-10 which bears no mutation, its binding to IL-2 is notaffected. Similarly mutations at position 125 and/or 127 do not affectbinding to mAbs H28 and 2C4, and serve as additional controls. ELISAexperiments performed with all the mutants support the data obtainedwith Western blots (data not shown).

mAb H2-8 (deposited at CNCM as accession no. I-2338 on, Oct. 21, 1999and 19B11 (described in Molec. Immunol. (1995) 32:1047-1056) havesimilar properties: both bind to the NH₂ terminal end of IL-2 andspecifically inhibit the binding of IL-2 to IL-2Rβ chain. Since bothantibodies recognize sequences located in peptide 1-30 it was ofinterest to compare the relationship between the corresponding epitopes.Plates coated with mAb H2-8 were used to bind peptide 1-30. The bindingof mAb 19B11 to these plates was positive, thus indicating that theepitopes of mAbs H2-8 and 19B11 do not overlap significantly. Variouscontrols performed to verify these results are shown (FIG. 3). Thebinding of 19B11 is strictly dependent on the presence of peptide 1-30and on the coating by mAb H2-8. Results obtained with mAb 3H9recognizing the peptide 30-54 further demonstrated the specificity ofthe data presented in FIG. 3.

Example 4

Cell Lines, Culture Media and Proliferation Assay

TS1 cells express only mouse IL-2Rγ. TS1β cells were obtained aftertransfection of TS1 cells with human IL-2Rβ cDNA cloned in the pdKCRexpression vector kindly provided by Dr. T. Taniguchi (Institute forMolecular and Cellular Biology, Tokyo University, Japan). TS1α cellswere obtained after transfection of TS1 cells with human IL-2Rα cDNAcloned in pCMV4 expression vector provided by Drs W. A. Kuziel and W. C.Greene (Gladstone Institute Virol./Immunol., San Francisco Calif., USA).TS1β and TS1α were previously characterized (Pitton et al. (1993)Cytokine 5:362-371). CTLL2 and YT were also used for IL-2 bindingstudies.

All cultures were performed in complete medium composed of RPMI 1640(BioProducts, Walkerville, Md.), 10% heat inactivated FCS (Serovial,Vogelgsun, France), 2 mM glutamine, 100 units/ml penicillin, 100 μg/mlstreptomycin, 50 mM 2-β-mercaptoethanol (2βME). TS1β and TS1α cell lineswere grown as TS1 cells in complete medium supplemented with supernatantof recombinant baculovirus expressing murine IL-9 proteins (DIB 349)(Uyttenhove et al. (1988) Proc. Natl. Acad. Sci. USA 85:6934-6938).

TS1β cells were cultured (10⁴ cells/well) in 96 wells in flat-bottomedmicrotiter plates with a final volume of 0.2 ml. Various concentrationsof human rIL-2, IL-2 muteins or mouse rIL-9 were assayed. In order totest the inhibitory effect of mAbs, different concentrations of thesereagents were mixed in the culture wells with the respective lymphokinesfor 30 min at low temperature before adding the cells. The inhibitoryeffects of mutein 20 Leu (described in Cytokine (1997) 9:488-498, whichis incorporated herein by reference in its entirety) was studied bypreincubating the cells (30 min at 4° C.) with the indicatedconcentration of inhibitor before adding IL-2 or IL-9 to the wells.Cultures were pulsed with 0.5 μCi/well of (³H) TdR after 36 h ofincubation and harvested 15 h later.

Example 5

Biological Properties of mAb H2-8

The biological properties of mAb H2-8 were evaluated on theproliferation of the IL-2- or IL-9 -dependent TS1β cell line (FIG. 4).In these experiments the IL-2 mutant IL-2 Pro¹²⁵ (Cys→Pro) was used.This mutation slightly reduces the affinity of IL-2 for IL-2R withoutaffecting the maximum proliferation obtained when higher concentrationsof mutant are used.

FIG. 4 shows that different concentrations of mAb H2-8 reduce the IL-2proliferation of TS1β. A progressive shift of the IL-2 titration curveis observed with increasing concentrations of mAb H2-8. Inhibitoryeffects of mAb H2-8 are comparable to those obtained with mAb 19B11,which was also found to inhibit the proliferation of cells bearing highaffinity IL-2R (Moreau et al. (1995b) Mol. Immunol. 32:1047-1056).Addition of both mAb H2-8 and 19B11 completely abolishes IL-2proliferation even at a very high dose of IL-2.

As controls FIG. 4 shows that mAb 11B11 (specific for mouse IL-4) doesnot affect the IL-2-dependent proliferation of TS1β. IL-9-inducedproliferation of TS1β is also not affected by either H2-8 or 19B 11.

Example 6

IL-2 Binding Assay and Inhibition

The IL-2 binding assay was performed as already described (Moreau et al.(1995b) Mol. Immunol. 32:1047-1056). ¹²⁵I-labelled IL-2 binding todifferent cell lines was first studied. Inhibition experiments wereperformed at concentration of ¹²⁵I-labelled IL-2 giving between 50 to70% maximum binding. The effects of the different muteins was analyzedafter 1 hr preincubation at 4° C. followed by incubation with¹²⁵I-labelled IL-2 (3 hr at 4° C.). In each experiments non-specificbinding was determined. The data were expressed as % inhibitory capacityof the different mutein versus wild type protein.

Example 7

Physico-chemical Properties of Peptide IP130 (Cytokine 1997)

The amino terminal peptide of IL-2 including aa 1 to 30 has a molecularweight of 3422.

The circular dichroism studies performed with IP130 indicates that at20° C., in phosphate buffer (20 mM, pH7.2), 50% of the residues are inan a helix configuration.

The quaternary structure of peptide IP130 was also studied bysedimentation-diffusion equilibrium. At concentration above 5×10⁻⁶ M,most of the molecules are in a tetrameric form (in equilibrium with anoctomeric complex).

The aminoacid sequence 1 to 30 shows 7 leucines and 2 isoleucines amongthe first 20 residues. The periodicity of these aa as well as the aboveresults suggest a structural model for IP130 that would comprise 4peptides organized in 4 α helices. In this model leucines andisoleucines side chains appear on the same face. This face ishydrophobic and four of these faces would build an hydrophobic coreinacessible to water. At high concentration peptide 1-10 tend todimerize and this would explain the formation of octameric peptides.

The binding of IP130 to soluble IL-2Rβ chain was studied. Soluble IL-2Rβhas been found to be dimeric in solution. From the results, a structuralmodel has been proposed: the complex would include four IP130 peptidesand two IL-2Rβ chains ((IP130)₄/(IL-2Rβ)₂).

Example 8

Biological Properties of IP130

Studies were performed either with a murine cell line transfected byhuman IL-2Rβ gene (TS1β) or with an IL-2 dependant human leukemic cellline (Kit 225 from Dr. T. Hori).

IP130 stimulates the proliferation of TS1β in the absence of IL-2. Inthe presence of IL-2 a strong synergy is observed with the peptide. Bothactivities are obtained at comparable concentrations (IC-50≅μM).

IP130 acts only on cell lines expressing human IL-2Rβ. This is inagreement with previous studies showing that murine IL-2Rβ does not bindIL-2 (Chastagner et al. 1996, Eur. J. immunol. 26:201-6). Consequently,classical murine cell lines (C30-1, CTLL, HT-2, . . . ) usually used toassay IL-2 activity remains insensitive to IP130 effects. Furthermoreanti-human IL-2Rβ blocking mAb neutralizes the effects of IP130.

Alone the peptide induces the phosphorylation of proteins on Kit 225cell line. On the pattern of phosphorylated proteins, the kinase Shc iseasily recognized. After specific immunoprecipitation and blotting withmAb 4G10 (anti-Ptyr), phosphorylated IL-2Rβ is identified on lysatesfrom kit 225 cell line stimulated by IP130. c-myc induction whichdepends on IL-2Rβ phosphorylation is also observed after IP130stimulation. STAT-3 and STAT-5 are not activated after IP130 stimulationsince IL-2Rγ is not involved in IP130 interaction with Kit 225.

Example 9

Immunological Properties of IP130 and use as Therapeutic Agent

IL-2Rβ chain is constitutively expressed by human NK cells from alldonors studied (David et al., Blood 91:165-172, 1998). Monocytes onlyexpress IL-2Rγ chain. Other lymphocytes do not express neither IL-2Rβnor IL-2Rα nor IL-2Rγ (David et al. 1998).

IP130 induces NK cells to enter into the cell cycle.

IP130 has been also tested on the generation of LAK in vitro. IP130induces LAK activity as tested on K562 targets.

Discussion

mAb H2-8 was isolated after immunization with IL-2 peptide 1-30. Itrecognizes both IL-2 and peptide 1-30, but not the shorter peptidescovering the same region (FIG. 2). This result suggests that mAb H2-8recognizes a conformational epitope on the N-terminal region of IL-2,and that this epitope is mimicked by the 1-30 peptide. Indeed, circulardichroism measurements reveal a significant fraction of α-helicalstructure for the 1-30 peptide. Furthermore, H2-8 can bind to peptide1-30 even in the presence of mAb 19B11 (which recognizes a linearepitope within the non-helical part of the peptide 1-30), but does notrecognize IL-2 mutants at position 20 (in the center of t-helix A) asdetermined by Western blot analysis or ELISA (FIG. 3 and other data notshown). The antibody also inhibits the bioactivity of IL-2 on TS1β cells(FIG. 4), whose proliferation is strictly dependent on the expression ofthe human IL-2Rβ chain. Taken together, these results demonstrate thatmAb H2-8 recognizes an epitope around Asp20 of IL-2, a region thatdirectly influences the interaction of the cytokine with IL-2Rβ.

Having now fully described the invention, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

3 1 93 DNA Artificial Sequence Synthetic DNA 1 atggctccga cgagcagctccaccaagaaa acccagctcc agctcgaaca cctgctgctg 60 gacctgcaga tgatcctgaacggtatcaac aac 93 2 31 PRT Artificial Sequence Synthetic Peptide 2 MetAla Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu 1 5 10 15His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 20 25 30 3132 PRT Artificial Sequence Synthetic Peptide 3 Ala Pro Thr Ser Ser SerThr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp LeuGln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu TyrArg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu LeuLys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu ValLeu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg AspLeu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser GluThr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val GluPhe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile 115 120 125 Ser ThrLeu Thr 130

What is claimed as new and is desired to be secured by Letters Patent ofthe United States is:
 1. An isolated DNA sequence encoding a peptideconsisting of the amino acid sequence set forth in SEQ ID NO:2.
 2. Avector comprising the DNA sequence of claim
 1. 3. The DNA sequence ofclaim 1, wherein said DNA sequence consists of the nucleic acid sequenceset forth in SEQ ID NO:1.
 4. The DNA sequence of claim 1, wherein saidDNA sequence is at least 90% homologous to SEQ ID NO:1.
 5. The vector ofclaim 2, wherein said DNA sequence is operatively linked to anexpression control sequence.
 6. The vector of claim 5, wherein saidexpression control sequence is one or more selected from the groupconsisting of an early SV40 promoter, a late SV40 promoter, an early CMVpromoter, a late CMV promoter, an early vaccinia promoter, a latevaccinia promoter, an early polyoma promoter, a late polyoma promoter,an early adenovirus promoter, a late adenovirus promoter, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage λ, the control regionsof fd coat protein, the promoter for 3-phosphoglycerate kinase, thepromoter of a glycolytic enzyme, the promoter of an acid phosphotase,and the promoter of the yeast α-mating factor.
 7. A unicellular hostcell transformed with the vector of claim
 2. 8. The unicellular hostcell of claim 7, wherein said unicellular host cell is a eukaryotic hostcell or an prokaryotic host cell.
 9. The unicellular host cell of claim7, wherein said unicellular host cell is a culture or tissue culture ofcells selected from the group consisting of E. coli, Pseudomonas,Bacillus, Streptomyces, a yeast, a CHO animal cell, a R1.1 animal cell,a B-W animal cell, a L-M animal cell, a COS 1 African Green Monkeykidney cell, a COS 7 African Green Monkey kidney cell, a BSC1 AfricanGreen Monkey kidney cell, a BSC40 African Green Monkey kidney cell, aBMT10 African Green Monkey kidney cell, a Sf9 insect cell, a murine TS1cell, a human cell, and a plant cell.
 10. An isolated DNA sequenceencoding a peptide consisting of an amino acid sequence that is at least90% homologous to the amino acid sequence set forth in SEQ ID NO:2. 11.The DNA sequence of claim 10, wherein said DNA sequence is at least 90%homologous to SEQ ID NO:1.
 12. A vector comprising the DNA sequence ofclaim
 10. 13. The vector of claim 12, wherein said DNA sequence isoperatively linked to an expression control sequence.
 14. The vector ofclaim 13, wherein said expression control sequence is one or moreselected from the group consisting of an early SV40 promoter, a lateSV40 promoter, an early CMV promoter, a late CMV promoter, an earlyvaccinia promoter, a late vaccinia promoter, an early polyoma promoter,a late polyoma promoter, an early adenovirus promoter, a late adenoviruspromoter, the lac system, the trp system, the TAC system, the TRCsystem, the LTR system, the major operator and promoter regions of phageλ, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase, the promoter of a glycolytic enzyme, thepromoter of an acid phosphate, and the promoter of the yeast α-matingfactor.
 15. A unicellular host cell transformed with the vector of claim12.
 16. The unicellular host cell of claim 15, wherein said unicellularhost cell is a eukaryotic host cell or an prokaryotic host cell.
 17. Theunicellular host cell of claim 15, wherein said unicellular host cell isa culture or tissue culture of cells selected from the group consistingof E. coli, Pseudomonas, Bacillus, Streptomyces, a yeast, a CHO animalcell, a R1.1 animal cell, a B-W animal cell, a L-M animal cell, a COS 1African Green Monkey kidney cell, a COS 7 African Green Monkey kidneycell, a BSC1 African Green Monkey kidney cell, a BSC40 African GreenMonkey kidney cell, a BMT10 African Green Monkey kidney cell, a Sf9insect cell, a murine TS1 cell, a human cell, and a plant cell.